Florida Institute of Technology.
Florida Institute of Technology;
J Vis Exp. 2024 Jul 12(209). doi: 10.3791/67141.
Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 °C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter "CaFE" for Calcium during Fertilization in C. elegans. The CaFE reporter was engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.
钙离子是卵母细胞到胚胎转变（OET）和早期胚胎发生过程中的重要信号分子。雌雄同体的秀丽隐杆线虫为 OET 的研究提供了几个独特的优势，因为它是透明的，并且具有有序的生殖腺，在 20°C 下每 23 分钟左右产生一个成熟的卵母细胞。我们已经对遗传编码的钙指示剂 jGCaMP7s 进行了修改，使其能够在活体生物中荧光指示受精的时刻。我们将这个报告器命名为“CaFE”，代表秀丽隐杆线虫受精过程中的钙离子。CaFE 报告器被工程改造到一个单拷贝的安全港基因座中，对生理或生育能力没有显著影响，并且在受精时产生强烈的信号。在这里，我们提出了一系列利用 CaFE 报告器作为体内工具来剖析 OET 和胚胎发生的方案。我们包括了同步蠕虫、检查 RNAi 敲低影响、为成像安装蠕虫以及在卵母细胞和胚胎中可视化钙离子的方法。此外，我们还介绍了生成其他蠕虫株系来辅助这种类型分析的方法。为了展示 CaFE 报告器在可视化受精时间方面的实用性，我们报告了当 ipp-5 被 RNAi 靶向时会发生双排卵，并且只有第一个卵母细胞会立即受精。此外，我们还报告了在早期胚胎发生过程中发现的单细胞钙瞬变，表明 CaFE 报告器在早期发育中仍然存在。重要的是，蠕虫中的 CaFE 报告器足够简单，可以用于纳入基于课程的本科研究（CURE）实验室课程。CaFE 报告器与线虫有序的生殖腺和易于进行 RNAi 相结合，有助于研究调节内部受精和早期胚胎发生所需的细胞间动力学。
